Overexpression, Purification, and Characterization of Glutaminase-Interacting Protein, a PDZ-Domain Protein from Human Brain

Aledo, Juan Carlos; Rosado, Abel; Olalla, Lucı́a; Campos, José A.; Márquez, Javier

doi:10.1006/prep.2001.1522

Cited by 15 publications

(15 citation statements)

References 27 publications

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“…It is known that the majority of PDZ proteins contain multiple protein–protein interaction domains, which act as molecular scaffolds important for a variety of cellular functions including cell signalling. The Tip-1 protein is known to form dimers in solution (Aledo et al , 2001) and it has been reported to be a negative regulator of PDZ-based scaffolding assemblies (Alewine et al , 2006). Therefore, it is possible that the binding of HPV16 E6 to the Tip-1 PDZ domain may affect its ability to interact with and regulate a signalling pathway involving GEF16 and Cdc42, resulting in an increase in activated Cdc42 levels.…”

Section: Discussionmentioning

confidence: 99%

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The HPV16 E6 binding protein Tip-1 interacts with ARHGEF16, which activates Cdc42

Oliver

Borthwick

et al. 2010

Br J Cancer

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Background:Guanidine exchange factor (GEF)-catalysed activation of Rho proteins such as Cdc42 has been shown to have a crucial role in cellular transformation, malignant progression and invasion. We have previously shown that the HPV16 E6 oncoprotein binds to the PDZ domain protein Tax-interacting-protein 1 (Tip-1) and we now report identification and functional analysis of a novel Tip-1 binding GEF.Methods:Yeast two-hybrid, in vitro pull-down, site-directed mutagenesis, semiquantitative PCR, co-immunoprecipitation and western blotting were used to identify/confirm novel Tip-1 binding partners and analyse cellular expression levels. In vitro kinetic analyses of recombinant proteins, siRNA gene silencing and in cell assays were used to measure Rho protein activation.Results:Tax-interacting-protein 1 was shown to interact with ARHGEF16 by its carboxyl PDZ binding motif. Levels of ARHGEF16 were increased in transformed and immortalised cells expressing ectopic HPV16 E6 and Cdc42 was co-immunoprecipitated by ARHGEF16 in the presence of high-risk HPV E6. In vitro kinetic analysis confirmed that recombinant ARHGEF16 activates Cdc42 and this was increased by the addition of recombinant Tip-1 and E6. Cells expressing HPV16 E6 had higher levels of Cdc42 activation, which was decreased by siRNA silencing of either Tip-1 or ARHGEF16.Conclusion:These data suggest that HPV16 E6, Tip-1 and ARHGEF16 may cooperate to activate Cdc42 and support a potential link between the expression of HPV16 E6 and Cdc42 activation.

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Section: Discussionmentioning

confidence: 99%

“…As Tip-1 has been shown to exist as a dimer in solution (Aledo et al , 2001), this raises the possibility that Tip-1 may form a link between E6 and components of these pathways. This would be consistent with the proposed ability of viral oncoproteins to promote transcriptional re-programming by modifying Rho and Wnt protein signalling.…”

mentioning

confidence: 99%

The HPV16 E6 binding protein Tip-1 interacts with ARHGEF16, which activates Cdc42

Oliver

Borthwick

et al. 2010

Br J Cancer

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“…In this context, a working hypothesis could be outlined. This is based upon the following circumstantial evidence: (i) the existence of a LXXLL motif in the N-terminal region of LGA, (ii) the ability of the C terminus of LGA to interact with PDZ domain-containing proteins (24), and (iii) the fact that this protein-protein interaction can affect glutaminase activity (14). Therefore, it is tempting to postulate that LGA may form part of a transcriptional complex as a coregulator and that the conversion of glutamine to glutamate, after glutaminase activation, may facilitate the association/dissociation to/from nuclear receptors, particularly in light of the evidence of rapid turnover of receptor interactions (28).…”

Section: Discussionmentioning

confidence: 99%

“…Cytochrome c oxidase and phosphate-activated glutaminase were assayed by standard enzymic methods (12,13). Western blotting was carried out essentially as described elsewhere (14), and the blots were developed with the enhanced chemiluminescence technique as recommended by the supplier (Amersham Biosciences).…”

Section: Methodsmentioning

confidence: 99%

Nuclear Localization of L-type Glutaminase in Mammalian Brain

Olalla¹,

Gutiérrez²,

Campos³

et al. 2002

Journal of Biological Chemistry

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In mammals, there are two different genes encoding for glutaminase isoforms, named liver (LGA) and kidney (KGA) types. LGA has long been believed to be present only in liver mitochondria from adult animals. However, we have recently reported the presence of LGA mRNA in human brain. We now describe the expression of LGA mRNA in the brain of other mammals (cow, mouse, rabbit, and rat) and in different areas of human brain as assessed by Northern blot analysis. The presence of mRNA encoding for this isoform in rat brain was further confirmed by reverse transcriptase-PCR cloning and sequencing. Although it has been well accepted that glutaminase is a mitochondrial enzyme, using newly generated isoform-specific antibodies, we have found a differential intracellular immunolocalization of both glutaminase isoforms in rat and monkey brain. In both species, KGA protein was present in mitochondria, whereas LGA protein was localized in nuclei. Furthermore, subcellular fractionation and Western blot analysis revealed that brain LGA was enriched in nuclei where it was catalytically active. Nuclear glutaminase exhibited a kinetic behavior that resembles that of the liver-type enzyme with regard to the low phosphate concentration requirement; however, nuclear glutaminase was susceptible to glutamate inhibition, a property that is absent in the rat liver enzyme.

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“…In fact, GIP has been shown to inhibit L-type GA activity in crude extracts of rat liver (71). Interestingly, a role in the targeting of PDZ protein interaction partners to concrete subcellular localizations, including cell nucleus, has also been reported (72).…”

Section: Other Ga Functions In Mammalian Brainmentioning

confidence: 99%

Brain glutaminases

Márquez

Martín-Rufián

Segura

et al. 2010

BioMolecular Concepts

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Glutaminase is considered as the main glutamate producer enzyme in brain. Consequently, the enzyme is essential for both glutamatergic and gabaergic transmissions. Glutaminederived glutamate and ammonia, the products of glutaminase reaction, fulfill crucial roles in energy metabolism and in the biosynthesis of basic metabolites, such as GABA, proteins and glutathione. However, glutamate and ammonia are also hazardous compounds and danger lurks in their generation beyond normal physiological thresholds; hence, glutaminase activity must be carefully regulated in the mammalian brain. The differential distribution and regulation of glutaminase are key factors to modulate the metabolism of glutamate and glutamine in brain. The discovery of novel isoenzymes, protein interacting partners and subcellular localizations indicate new functions for brain glutaminase. In this short review, we summarize recent findings that point consistently towards glutaminase as a multifaceted protein able to perform different tasks. Finally, we will highlight the involvement of glutaminase in pathological states and its consideration as a potential therapeutic target.

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Overexpression, Purification, and Characterization of Glutaminase-Interacting Protein, a PDZ-Domain Protein from Human Brain

Cited by 15 publications

References 27 publications

The HPV16 E6 binding protein Tip-1 interacts with ARHGEF16, which activates Cdc42

The HPV16 E6 binding protein Tip-1 interacts with ARHGEF16, which activates Cdc42

Nuclear Localization of L-type Glutaminase in Mammalian Brain

Brain glutaminases

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